ObjectiveTo investigate the antitumor effect and mechanism of Guiqi Yiyuan ointment on Lewis lung cancer mice based on the extracellular regulatory protein kinase （ERK） signaling pathway. MethodsA Lewis lung cancer mouse model was established. Except for the blank group， the model mice were randomly divided into the model group， Guiqi Yiyuan ointment low， medium， and high dose groups， and the extracellular ERK1/2 inhibitor group， with 10 mice per group. The Guiqi Yiyuan ointment was administered by gavage at doses of 1.75， 3.5， 7.0 g·kg^-1·d^-1 for the low， medium， and high dose groups， respectively. The ERK1/2 inhibitor group was given the ERK1/2 inhibitor LY3214996 （100 mg·kg^-1·d^-1） by gavage. The treatment was administered for 14 consecutive days， after which samples were collected. Tumor histopathological changes were observed using hematoxylin-eosin （HE） staining. Transmission electron microscopy was used to observe ultrastructural changes in tumor cells. Immunofluorescence was performed to measure the phosphorylation of ERK1/2 （p-ERK1/2） and the expression of programmed cell death ligand-1 （PD-L1） in tumor tissues. Western blot and real-time quantitative polymerase chain reaction （Real-time PCR） were used to detect the expression of p-ERK1/2， PD-L1， the autophagy marker Beclin-1， the autophagic protein p62， and the microtubule-associated protein light chains LC3Ⅰ and LC3Ⅱ at both the protein and gene levels. ResultsCompared with the model group， the average tumor weight was significantly reduced in the low and medium dose groups of Guiqi Yiyuan ointment （P<0.05）， and markedly reduced in the high dose and inhibitor groups （P<0.01）. Tumor cells in all treatment groups became progressively irregular， with ruptured nuclei and expanded areas of cell disintegration and necrosis. The number of organellar ablations in tumor tissues increased， and the number of autophagic vesicles also increased in all groups. The mean fluorescence intensity of p-ERK1/2 and PD-L1 was reduced in the low and medium dose groups of Guiqi Yiyuan ointment （P<0.05）， and significantly reduced in the high dose and inhibitor groups （P<0.01）. The mRNA expression of ERK1/2， PD-L1， Beclin-1， and p62 was reduced in the medium dose group （P<0.05）， while LC3Ⅰ/Ⅱ mRNA expression was elevated （P<0.05）. In the high dose and inhibitor groups， mRNA expression of ERK1/2， PD-L1， Beclin-1， and p62 was significantly reduced （P<0.01）， while LC3Ⅰ/Ⅱ mRNA expression was significantly increased （P<0.01）. Protein expression of p-ERK1/2， PD-L1， Beclin-1， and p62 was reduced in the medium dose group （P<0.05）， and LC3Ⅰ/Ⅱ protein expression was elevated （P<0.05）. In the high dose and inhibitor groups， protein expression of p-ERK1/2， PD-L1， Beclin-1， and p62 was significantly reduced （P<0.01）， while LC3Ⅰ/Ⅱ protein expression was significantly elevated （P<0.01）. ConclusionGuiqi Yiyuan ointment may inhibit the activation of the ERK signaling pathway， downregulate the expression of p-ERK1/2， promote autophagic flux in tumor cells， and regulate the expression of PD-L1， thereby exerting an inhibitory effect on tumor growth in Lewis lung cancer mice.

This study aims to investigate the anti-tumor effect and mechanism of Guiqi Yiyuan Ointment combined with cisplatin on Lewis lung carcinoma-bearing mice via the protein kinase RNA-like endoplasmic reticulum kinase(PERK)/eukaryotic translation initiation factor 2α(eIF2α)/activated transcription factor 4(ATF4)/C/EBP homologous protein(CHOP) signaling pathway. Sixty SPF-grade male C57BL/6 mice were selected and assigned into a blank group and a modeling group by the random number table method. After modeling of the Lewis lung carcinoma, the mice in the modeling group were randomized into model, cisplatin(5 mg·kg~(-1), once a week), and low-, medium-, and high-dose(1.7, 3.5, and 7.05 g·kg~(-1), respectively, once a day) Guiqi Yiyuan Ointment+cisplatin(5 mg·kg~(-1)) groups(n=10). After 14 days of continuous intervention, the spleen, thymus, and tumor samples of the mice were collected, weighed, and recorded, and the spleen index, thymus index, and tumor suppression rate were calculated. Hematoxylin-eosin(HE) staining was employed to observe the pathological changes in the tumor tissue. The morphological changes of the endoplasmic reticulum of tumor cells were observed by transmission electron microscopy. The positive expression of phosphorylated eIF2α(p-eIF2α) and ATF4 in the tumor tissue was detected by immunofluorescence. Western blot was employed to determine the protein levels of phosphorylated PERK(p-PERK), p-eIF2α, ATF4, CHOP, B-cell lymphoma-2(Bcl-2), Bcl-2-associated X protein(Bax), cyclin-dependent kinase inhibitor 1A(p21), and cyclinD1 in the tumor tissue. Real-time fluorescent quantitative PCR was employed to determine the mRNA levels of PERK, eIF2α, ATF4, CHOP, Bax, Bcl-2, p21, and cyclinD1 in the tumor tissue. Compared with the blank group, the model group showed decreases in spleen index and thymus index(P<0.05). Compared with the model group, the cisplatin group showed decreases in spleen index and thymus index(P<0.05), and the medium-and high-dose Guiqi Yiyuan Ointment+cisplatin groups presented increases in spleen index and thymus index(P<0.05). In addition, the treatment groups all showed decreased tumor mass(P<0.05), increased tumor cell lysis and nuclear rupture, widened gap between rough endoplasmic reticulum, enhanced average fluorescence intensity of p-eIF2α and ATF4(P<0.05), up-regulated protein levels of p-PERK/PERK, p-eIF2α/eIF2α, ATF4, CHOP, Bax, and p21(P<0.05), down-regulated protein and mRNA levels of Bcl-2 and cyclinD1(P<0.05), and up-regulated mRNA levels of PERK, eIF2α, ATF4, CHOP, Bax, and p21(P<0.05). Compared with the cisplatin group, the combination groups showed increases in spleen index and thymus index(P<0.05) as well as mean optical density(P<0.05), and the high-dose Guiqi Yiyuan Ointment+cisplatin group showed decreased tumor mass(P<0.05). In addition, the medium-and high-dose Guiqi Yiyuan Ointment+cisplatin groups showcased enhanced average fluorescence intensity of p-eIF2α and ATF4(P<0.05), up-regulated protein levels of p-PERK/PERK, p-eIF2α/eIF2α, ATF4, CHOP, Bax, and p21(P<0.05), down-regulated protein and mRNA levels of Bcl-2 and cyclinD1(P<0.05), and up-regulated mRNA levels of PERK, eIF2α, ATF4, CHOP, Bax, and p21(P<0.05). In conclusion, Guiqi Yiyuan Ointment combined with cisplatin can effectively inhibit the growth of Lewis lung carcinoma in mice by regulating the expression of proteins related to the PERK/eIF2α/ATF4/CHOP signaling pathway and promoting cell cycle arrest and apoptosis.
Animals ; Cisplatin/administration & dosage* ; Activating Transcription Factor 4/genetics* ; Eukaryotic Initiation Factor-2/genetics* ; eIF-2 Kinase/genetics* ; Carcinoma, Lewis Lung/pathology* ; Drugs, Chinese Herbal/administration & dosage* ; Male ; Mice ; Signal Transduction/drug effects* ; Mice, Inbred C57BL ; Transcription Factor CHOP/genetics* ; Ointments/administration & dosage* ; Humans ; Cell Proliferation/drug effects* ; Antineoplastic Agents/administration & dosage*

BACKGROUND: Early control of low-density lipoprotein cholesterol (LDL-C) is crucial for reducing the progress of cardiovascular disease. However, its additional role to the risk of primary osteoporosis in men with coronary heart disease was inconclusive. Our study aims to determine the association of LDL-C and its trajectories for osteoporosis risk in the middle-aged and aged men of China. METHODS: The retrospective cohort study of 1546 men aged 69.74 ± 11.30 years conducted in Beijing, China from 2015 to 2022. And the incidence of primary osteoporosis was annually recorded. LDL-C trajectories were further identified by latent class growth model using repeated measurements of LDL-C. The association of baseline LDL-C for osteoporosis was estimated using hazard ratio (HR) with 95% CI in Cox proportional hazard model, while mean level and trajectories of LDL-C for osteoporosis were evaluated using odds ratio (OR) with 95% CI in logistic regression model. RESULTS: During the median 6.2-year follow-up period, 70 men developed primary osteoporosis. The higher level of baseline LDL-C (HR = 1.539, 95% CI: 1.012-2.342) and mean LDL-C (OR = 2.190, 95% CI: 1.443-3.324) were associated with higher risk of osteoporosis in men with coronary heart disease after adjusted for covariates. Compared with those in the LDL-C trajectory of low-stable decrease, participants with medium-fluctuant trajectory, whose longitudinal LDL-C started with a medium LDL-C level and appeared an increase and then decrease, were negatively associated with osteoporosis risk (OR = 2.451, 95% CI: 1.152-5.216). And participants with initially high LDL-C level and then a rapid decrease demonstrated a tendency towards reduced risk (OR = 0.718, 95% CI: 0.212-2.437). CONCLUSIONS Elevated LDL-C level and its long-term fluctuation may increase the risk of primary osteoporosis in men. Early controlling a stable level of LDL-C is also essential for bone health.

OBJECTIVE: To explore the genetic etiology of fetal skeletal dysplasia using whole exome sequencing (WES) and copy number variation sequencing (CNV-seq) techniques, and the feasibility of using WES as the first-tier method for such fetuses. METHODS: Seventy four fetuses with skeletal dysplasia detected by prenatal ultrasound at the Genetic Testing Center of the Women and Children's Hospital Affiliated to Qingdao University from January 2020 to August 2024 were selected as the study subjects. Fetal muscle and peripheral blood samples of the pregnant women and their spouses were collected and subjected to WES analysis. CNV-seq was carried out on all fetal muscle tissue samples. And the results were compared with the CNVs indicated by WES. Genetic etiologies were analyzed across different subtypes of skeletal dysplasia. And the feasibility of using WES as the first-tier genetic test for similar fetuses was assessed, in addition with a systematic cost-effectiveness analysis. This study was approved by the Medical Ethics Committee of the Hospital (Ethics No.: QFELL-YJ-2024-201). RESULTS: A total of 50 fetuses were diagnosed, which yielded a diagnostic rate of 67.57%. These included 6 chromosomal aneuploidies, 4 chromosomal CNVs and 40 monogenic disorders. The monogenic diseases had involved 46 variant sites in 23 pathogenic genes, among which 12 were unreported previously, including MYH3: c.735T>C, ALPL: c.1324C>T, NEK9: c.1973G>A, MAGEL2: c.2024_2025del, LMBR1: c.423+4914A>C, NEB: c.21273_21276del, COL1A1: c.2651G>C and c.2758G>C, ASPM: c.2473delinsGA, TBX5: c.704G>A, DYNC2H1: c.10893del, and DYNC2I2: c.1270C>T. Substantial concordance was reached between WES-derived CNV calls and CNV-seq findings. No clinically significant CNV was exclusively detected by CNV-seq. Cost-effectiveness modeling demonstrated that implementing WES as the first-tier genetic testing method could reduce the total expenditure when WES unit cost remained below 6.4 folds that of the CNV-seq. CONCLUSION Genetic variants including single nucleotide variations (SNV) of monogenic disorders, chromosomal aneuploidies and genomic CNVs are important causes for fetal skeletal dysplasia. WES is an accurate and efficient method for analyzing the etiology of fetal skeletal dysplasia, particularly in those with a family history of similar phenotype or maternal history of adverse pregnancies.
Humans ; Exome Sequencing/methods* ; Female ; Pregnancy ; DNA Copy Number Variations/genetics* ; Genetic Testing/methods* ; Prenatal Diagnosis/methods* ; Adult ; Male ; Fetus ; Bone Diseases, Developmental/diagnosis* ; Ultrasonography, Prenatal

OBJECTIVE: To explore the detection rate of copy number variations (CNVs) in fetuses with isolated Congenital anomalies of the kidney and urinary tract (CAKUT) and pregnancy outcomes in order to provide a basis for genetic counseling. METHODS: One hundred and eighty eight fetuses who underwent chromosomal microarray analysis (CMA) due to isolated CAKUT detected by prenatal ultrasonography at Qingdao Women and Children's Hospital from January 2021 to December 2024 were selected as the study subjects. According to the ultrasound findings, the fetuses were divided into 8 groups, including renal parenchymal dysplasia group, renal cystic dysplasia group, simple renal parenchymal echo enhancement group, abnormal development of renal collecting system group, duplicated kidney group, ectopic kidney group, horseshoe kidney group, and bladder/posterior urethral abnormalities group. The detection of CNVs was retrospectively analyzed, and the pregnant women were followed up to summarize their pregnancy outcomes. 2 test (or Fisher's exact probability method) was used to compare the CNV detection rates between the groups. This study was approved by the Medical Ethics Committee of the Qingdao Women and Children's Hospital (Ethics No.: QFELL-YJ-2025-85). RESULTS: Among the 188 fetuses with isolated CAKUT, 23 CNVs (12.23%) were detected, of which 13 cases (6.91%) were pathogenic and 10 cases were rated as variants of unknown significance (VOUS). Among the 8 groups, the three groups with the highest proportion were renal cystic dysplasia group, renal metaplasia group, and renal parenchymal dysplasia group. The detection rates of pathogenic CNVs in the three groups were 1.79% (1/56), 6.78% (4/59), and 16.67% (5/30), respectively, with statistically significant differences (P < 0.05). Parental verification was conducted on 12 fetuses detected with the CNVs, confirming that 2 cases were de novo and 10 were inherited from parents with a normal phenotype. After genetic counseling, the parents of 9 fetuses opted to terminate the pregnancy, while 11 chose to continue with the pregnancy, and 3 were lost to follow-up. At the time of last follow-up, the youngest offspring was 5 months old and the oldest was 3 years and 11 months old. One child had renal aplasia, and two were born with hydronephrosis, which have been cured through surgery. The remainders had no obvious abnormality with their growth and development. CONCLUSION CMA testing has important value for prenatal diagnosis of isolated CAKUT. In this study, the detection rate of pathogenic CNVs has increased sequentially in fetuses with renal cystic developmental abnormalities, renal collecting system developmental abnormalities, and renal parenchymal dysplasia, while there was no significant difference in the detection rate of CNVs. For fetuses with isolated CAKUT detected by prenatal ultrasound, CMA testing should be considered, and reasonable pregnancy decisions should be made based on the results of prenatal ultrasound and parental verification.
Humans ; Female ; Pregnancy ; Prenatal Diagnosis/methods* ; DNA Copy Number Variations/genetics* ; Kidney/abnormalities* ; Adult ; Ultrasonography, Prenatal ; Urogenital Abnormalities/diagnosis* ; Microarray Analysis/methods* ; Retrospective Studies ; Urinary Tract/abnormalities* ; Fetus ; Pregnancy Outcome ; Vesico-Ureteral Reflux

Objective To investigate the effect of cognitive-motor dual-task training on balance and gait in patients within six months post stroke. Methods From May,2022 to April,2023,68 stroke patients in Beijing Puren Hospital were randomly divided into con-trol group(n=34)and experimental group(n=34).Both groups received conventional rehabilitation training.The control group received single-task walking training,and the experimental group received cognitive-walking dual-task training,in addition,for four weeks.They were assessed with Fugl-Meyer Assessment-Lower Extremi-ties(FMA-LE)before and after training,and measured static balance posture trajectory,stability limits,gait anal-ysis with Pro-kin and OptoGait in both single-task and dual-task states. Results After training,the scores of FMA-UE increased in both groups(|t|>10.239,P<0.001),and no difference was found between two groups(P>0.05).In single-task state,all the parameters of balance improved in both groups after training(|t|>2.934,P<0.01),and the length of movement locus with eyes closed was longer in the experi-mental group than in the control group(t=3.330,P=0.001);all the parameters of gait improved in both groups after training(|t|>6.823,P<0.05),and the walk speed was better in the experimental group than in the control group(t=2.355,P=0.022).In dual-task state,all the parameters of balance improved in both groups after train-ing(|t|>2.472,P<0.05),expect the length of trajectory with eyes open in the control group(P>0.05),and no difference was found in other parameters between two groups(P>0.05);and all the parameters of gait improved in both groups after training(|t|>2.238,P<0.05),and the stride length and walk speed were better in the experi-mental group than in the control group(t>2.443,P<0.05). Conclusion Cognitive-motor dual-task training can effectively improve lower limb motor function,balance function and walking ability in convalescent stroke patients,and it is superior to single-task walking training in improving some gait parameters.

Objective:To explore the genetic etiology for a Chinese pedigree affected with Treacher-Collins syndrome (TCS) through whole exome sequencing (WES).Methods:A TCS pedigree which was diagnosed at the Women and Children′s Hospital Affiliated to Qingdao University on February 5 2020 was selected as the study subject. Following collection of clinical data, WES was carried out. Candidate variant was validated through Sanger sequencing and bioinformatic analysis..Results:The WES results showed that the proband has harbored a heterozygous c. 3337C>T variant of the TCOF1 gene, and Sanger sequencing confirmed that his mother and brother also carried the same variant. Based on guidelines from the American College of Medical Genetics and Genomics (ACMG), the variant was predicted as pathogenic (PVS1+ PM2_Supporting+ PP4). Conclusion:The heterozygous c. 3337C>T variant of the TCOF1 gene probably underlay the pathogenesis of TCS in this pedigree.

italic>Artemisia argyi is a traditional Chinese medicine in China, which is used as medicine with its leaves. The leaves of A. argyi mainly contain flavonoids, phenolic acids, volatile oils and other compounds, and have a variety of pharmacological activities. AP2/ERF transcription factors are abundant in plants and are mainly involved in plant growth and development, abiotic stress response and secondary metabolite biosynthesis regulation. However, there are few reports on the AP2/ERF gene family and its functions in A. argyi. In this study, we systematically identified the AP2/ERF gene family in A. argyi genome, and analyzed its phylogenetic tree, protein physicochemical properties, subcellular localization, conserved motifs, promoter elements, and expression patterns. The results showed that a total of 204 AP2/ERF transcription factors were identified in A. argyi genome, encoding proteins consisting of 88-483 amino acids with a relative molecular mass of 10-52.94 kDa and a theoretical isoelectric point of 4.62-9.88. Subcellular prediction showed that the majority of AP2/ERFs were located in the nucleus, cytoplasm, and the minority of them is located in the membrane and chloroplasts. According to the Arabidopsis AP2/ERF family classification, A. argyi AP2/ERF proteins were divided into four subfamilies: Soloist, AP2, ERF (B3, B4, B5, B6), and DREB (A1, A2, A4, A5, A6), among which DREB family accounted for the largest proportion, and the same subfamily had similar conserved motifs. cis-Acting element analysis showed that AP2/ERF promoters have a large number of elements responding to light and abiotic stress. Expression pattern analysis showed that most of the genes of the AP2/ERF family were dominantly expressed mainly in roots and stems, of which 19 were dominantly expressed in leaves, 77 would be induced to be expressed by methyl jasmonate, of which 16 genes were both dominantly expressed in leaves and induced to be expressed by methyl jasmonate, and these AP2/ERF genes may be the key regulatory genes for the synthesis of active ingredients in A. argyi leaves.This study lays the foundation for the functional study of AP2/ERF family genes and their regulating roles in the active components biosynthesis in A. argyi leaves.

Osteoarthritis(OA),characterized by cartilage degeneration,synovial inflammation,and subchondral bone remodeling,is among the most common musculoskeletal disorders globally in people over 60 years of age.The initiation and progression of OA involves the abnormal metabolism of chondrocytes as an important pathogenic process.Cartilage degeneration features mitochondrial dysfunction as one of the important causative factors of abnormal chondrocyte metabolism.Therefore,maintaining mitochondrial homeostasis is an important strategy to mitigate OA.Mitophagy is a vital process for autophagosomes to target,engulf,and remove damaged and dysfunctional mitochondria,thereby maintaining mitochondrial homeostasis.Cumulative studies have revealed a strong association between mitophagy and OA,suggesting that the regulation of mitophagy may be a novel therapeutic direction for OA.By reviewing the literature on mitophagy and OA published in recent years,this paper elaborates the potential mechanism of mitophagy regulating OA,thus providing a theoretical basis for studies related to mitophagy to develop new treatment options for OA.

The endoplasmic reticulum is a key site for protein production and quality control.More than one-third of proteins are synthesized and folded into the correct three-dimensional conformation in the endoplasmic reticulum.However,during protein folding,unfolded and/or misfolded proteins are prone to occur,which may lead to endoplasmic reticulum stress.Organisms can monitor the quality of the proteins produced by endoplasmic reticulum quality control(ERQC)and endoplasmic reticulum-associated degradation(ERAD),which maintain endoplasmic reticulum protein homeostasis by degrading abnormally folded proteins.The underlying mechanisms of protein folding and ERAD in mammals have not yet been fully explored.Therefore,this paper reviews the process and function of protein folding and ERAD in mammalian cells,in order to help clinicians better understand the mechanism of ERAD and to provide a scientific reference for the treatment of diseases caused by abnormal ERAD.